Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in <Emphasis Type="Italic">E. coli</Emphasis> and isolated by metal affinity chromatography

Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni²⁺-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni²⁺ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.     

Association of polymorphic markers of chemokine genes,their receptors,and <Emphasis Type="Italic">CD14</Emphasis> gene with coronary atherosclerosis

Atherosclerosis represents an inflammatory response to the disturbance of the endothelial layer in the arterial bloodstream. In the present study, an analysis of associations of polymorphic markers for the genes controlling synthesis of proteins involved in atherosclerosis pathogenesis in coronary atherosclerosis (CA) patients (217 subjects) and in a control group (250 subjects) was conducted. The following genes were examined: rs991804 (CCL2 gene), rs1126579 (CXCR2 gene), rs4074 (CXCL1 gene), rs4073 (CXCL8 gene), rs333 (CCR5 gene), rs2471859 (CXCR4 gene), rs1801157 (CXCL12 gene), and rs2569190 (CD14 gene). Using the Monte Carlo and Markov chain (APSampler) method, allele/genotype combinations associated with both low and high CA risk were revealed. The most important findings included the following: CXCR4*T/T + CCL2*C + CCR5*I/I (P_perm = 1 × 10^–6, OR = 0.44, 95% CI 0.3–0.63), CXCR2*C + CD14*C + CXCL12*G + CCL2*C + CCR5*D (P_perm = 4 × 10^–6, OR = 5.78, 95% CI 2.34–14.28), CD14*C + CCL2*C/C + CCR5*D (P_perm = 6.3 × 10^–6, OR = 5.81, 95% CI 2.17–15.56), CXCL8*A + CXCR2*C + CD14*T + CXCR4*C (P_perm = 0.01, OR = 3.21, 95% CI 1.63–6.31).     

Endonuclease Activity of MutL Protein of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> Mismatch Repair System

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C–terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N–terminal domain. rsMutL demonstrated the highest activity in the presence of Mn²⁺. The extent of plasmid DNA hydrolysis declined in the row Mn²⁺ > Co²⁺ > Mg²⁺ > Cd²⁺; Ni²⁺ and Ca²⁺ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn²⁺ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.     

Consortium of Higher Aquatic Plants and Microalgae Designed to Purify Sewage of Heavy Metal Ions

We selected higher aquatic plants (HAP) and microalgae possessing a high sorption capacity in respect to heavy metals to form a consortium designed to purify contaminated aquatic ecosystems. Accumulation of heavy metals Cd²⁺, Cu²⁺, Pb²⁺, and Zn²⁺ was investigated in plants Pistia stratiotes, Elodea canadensis, and Lemna minor and green microalgae Chlorella vulgaris ВВ-2, Ankistrodesmus sp. ВI-1, Chlamydomonas reinhardtii В-4, and Scеnеdеsmus quadricauda В-1. It was found that intense accumulation of metals occurs in cultures of HAP Pistia stratiotes and Elodea canadensis. These plants are macroconcentrators of zinc, lead, and copper and microconcentrators of cadmium. Out of the examined cultures of microalgae, effective bioaccumulators of heavy metals were C. vulgaris ВВ-2 and Ankistrodesmus sp. ВI-1. It was shown that heavy metals are selectively taken up from the medium in the series Zn²⁺ > Cu²⁺ > Cd²⁺ > Pb²⁺. In order to produce a consortium of higher aquatic plants and microalgae for purification of polluted aquatic ecosystems, we investigated interaction of HAP P. stratiotes and E. canadensis with microalgae C. vulgaris ВВ-2 and Ankistrodesmus sp. ВI-1 in the course of their cocultivation. Neutral relations were detected between the cells of microalgae C. vulgaris ВВ-2 and Ankistrodesmus sp. ВI-1 and HAP E. canadensis. At the same time, the cells of Ankistrodesmus sp. ВI-1 and HAP P. stratiotes formed a symbiosis. Microscopic examination showed numerous points where the cells of microalgae Ankistrodesmus sp. ВI-1 were attached to the roots of P. stratiotes plants. We tested an opportunity to employ the association between P. stratiotes and Ankistrodesmus sp. ВI-1 for purification of simulated wastewater polluted with heavy metal ions. This consortium proved to be capable of eliminating contaminants from the sewage, reducing their level in the sewage to standard values, and active accumulation of heavy metal ions.     

Cloning,purification and characterization of a cellulase-free xylanase from <Emphasis Type="Italic">Geobacillus thermodenitrificans</Emphasis> AK53

Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed K_M value to be 4.34 mg/mL (for D-xylose) and V_max value to be 2028.9 μmoles mg^–1 min^–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg²⁺ and Mn²⁺ were found to be required for the enzyme activity, however, Co²⁺, Hg²⁺, Fe²⁺ and Cu²⁺ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from<Emphasis Type="Italic"> Acidaminococcus fermentans</Emphasis>

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S]²⁺ cluster, 1.0 reduced riboflavin-5′-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S]^1+/2+-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg²⁺-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S]^1+/2+-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as ?405 mV and ?340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7–45 μM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 μmol/g protein at 7 μM Fe to 0.02 μmol/g at 45 μM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 μmol/g at 7–13 μM Fe to 1.1±0.1 μmol/g at 17–45 μM Fe.     

Dependence of expression of regulatory master genes of embryonic development in pancreatic cancer cells on the intracellular concentration of the master regulator PDX1

Exogenous expression of the gene encoding the pancreatic master regulator PDX1 in cell lines with different degrees of differentiation of pancreatic cancer cells is accompanied by changes in the expression of known master genes involved in cancer progression. In BxPC3^PDX+ cells, as compared to BxPC3^PDX–, we detected an increased expression of the following genes: NKX6.1 (2 times), NR5A2 (2.5 times), KLF5 (1.8 times), ZEB1 (3 times), and ONECUT1 (1.3 times), as well as a decreased expression of MUC1 and SLUG genes (3 and 2 times, respectively). In PANC1^PDX+ cells, as compared to the control PANC1^PDX– cells, we detected a decreased expression of ISL1 (2 times) and an increased expressed of KRT8 (2 times) and MUC1 (by 30%). In the high-grade cell lines (including the BxPC3 line studied), the total content of sites containing the marks of active enhancers was higher than that in the low-grade cell lines (PANC1).     

Isolation,expression, and functional analysis of developmentally regulated plasma membrane polypeptide 1 (DREPP1) in <Emphasis Type="Italic">Sporobolus virginicus</Emphasis> grown under alkali salt stress

The plant specific DREPP proteins have been shown to bind Ca²⁺ and regulate the N-myristoylation signaling and microtubule polymerization in Arabidopsis thaliana. The information about DREPP proteins in other plants is, however, scarce. In the present study, we isolated the DREPP gene from a halophytic grass, Sporobolus virginicus, and tested whether the gene was involved in alkaline salt stress responses. The SvDREPP1 was cloned from S. virginicus by RACE methods. The isolated gene showed high homology to DREPP homologs from C₄ grasses, Setaria italica, and Panicum hallii as well as rice (OsDREPP1). The encoded protein contained 202 amino acid residues. It was expressed in E. coli, and its biochemical properties were studied. It was observed that SvDREPP1 was not only Ca²⁺-binding protein, but also bind to calmodulin and microtubules. The SvDREPP1 mRNA expression in plants grown under alkaline salt stress was upregulated by 3.5 times over the control in leaf tissues after 48-h treatment, whereas it was increased for 6.0 times in the root tissues at 36 h. The data suggests the importance of SvDREPP1 in regulating alkali salt stress responses in the leaf tissues.     

Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

<Emphasis Type="Italic">Limnobacter humi</Emphasis> sp. nov., a thiosulfate-oxidizing,heterotrophic bacterium isolated from humus soil,and emended description of the genus <Emphasis Type="Italic">Limnobacter</Emphasis> Spring <Emphasis Type="Italic">et al.</Emphasis> 2001

Three Gram-negative, strictly aerobic, chemolithoheterotrophic bacterial strains, designated UCM-30, UCM-33, and UCM-39^T, were isolated in South Korea. Based on their 16S rRNA gene sequences, the three isolated strains were found to be similar to Limnobacter thiooxidans CS-K2^T (97.41–97.68%), Limnobacter litoralis KP1-19^T (95.55–95.76%), and various genera belonging to the class Betaproteobacteria (90.34–93.34%). DNA-DNA hybridization showed 79.3–83.9% similarity between the genomic DNA of UCM-39^T, UCM-30, and UCM-33, while the sequence similarity between UCM-39^T and L. thiooxidans KACC 13837T or L. litoralis LMG 24869T was 23.7% and 18.6%, respectively. The DNA G+C content of UCM 39T was 59.7 mol%, the major ubiquinone was Q-8, and the optimal oxidation rate was observed at 10 mM thiosulfate. The major fatty acids (≥ 10%) were summed features 3 (C_16:1 ω7c and/or C_16:1 ω6c) and 8 (C_18:1 ω7c and/or C_18:1 ω6c), and C_16:0. The major polar lipids (diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol) were found in all members of genus Limnobacter. Based on phenotypic, physiological, and phylogenetic analyses, the UCM-39T strain was found to be significantly distinct to represent a novel species affiliated to the genus Limnobacter. We propose to name it Limnobacter humi sp. nov. with the type strain UCM-39^T (=KACC 18574^T =NBRC 111650^T).     

Recombinant production of a shell matrix protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application to the biomimetic synthesis of spherulitic calcite crystals

Objectives

To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO₃ biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized.

Results

A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l^?1 with a purity of >95 %. It efficiently formed a complex with Ca²⁺. Ca²⁺-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20–30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein.

Conclusions

Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.

     

Karyotypic study of five Lutjanid species using conventional and Ag-NORs banding techniques

The first cytogenetic comparisons of five snapper species from Thailand were presented here. Renal cell samples were taken from blacktail snapper (Lutjanus fulvus), five lined snapper (L. quinquelineatus), dory snapper (L. fulviflamma), brownstripe red snapper (L. vitta), and mangrove red snapper (L. argentimaculatus). The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results exhibited that all five snapper species have the diploid chromosome numbers of 2n = 48 and the fundamental numbers (NF) of 48. The presences of large, medium, and small telocentric chromosomes were 22-24-2, 24-20-4, 36-10-2, 28-16-4 and 36-10-2, respectively. The Ag- NORs banding technique provides the pair of nucleolar organizer regions (NORs) at subcentromeric region of the long arm of the respective telocentric chromosome pairs 9, 1, 3, 4 and 9. Their karyotype formulas is as follows: L. fulvus (2n = 48): L ₂₂ ^t + M ₂₄ ^t + S ₂ ^t , L. quinquelineatus (2n = 48): L ₂₄ ^t + M ₂₀ ^t + S ₄ ^t , L. fulviflamma (2n = 48): Lt36 + Mt10 + St2, L. vitta (2n = 48): L ₂₈ ^t + M ₁₆ ^t + S ₄ ^t , and L. argentimaculatus (2n = 48): L ₃₆ ^t + M ₁₀ ^t + S ₂ ^t .     

Colony formation of highly dispersed <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> by controlling extracellular polysaccharides and calcium ion concentrations in aquatic solution

This study isolated extracellular polysaccharides (EPS) as a powder material from cyanobacterial blooms and the powdered EPS was used to trigger colony formation of dispersed unicellular M. aeruginosa by controlling EPS concentration in culture medium. The effect of Ca²⁺ ions on the colony formation of M. aeruginosa was also investigated, then the interaction between EPS and Ca²⁺ ions on colony formation was discussed. The results showed that the addition of the powdered EPS into the medium did not cause morphological changes of M. aeruginosa, suggesting that EPS alone would not induce the colony formation of M. aeruginosa. On the other hand, a high concentration of calcium ions (1000 mg/l) caused colony formation. When EPS and Ca²⁺ ions in the culture medium were adjusted to 200 and 1000 mg/l, respectively, the colony density, the average cell number per colony and the particle size of M. aeruginosa showed ca. 1.7–2.0 times greater values than those in the Ca²⁺ added medium. Calcium ion contributed to the aggregation of M. aeruginosa via crosslinked reaction with negatively charged M. aeruginosa cells, and the addition of EPS possessing negatively charged functional groups such as carboxy groups could enhance the reaction, promoting the crosslinked reaction between EPS and Ca²⁺ ions.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.